The principal reservoirs of Vibrio cholerae O1 in nature are rivers, estuaries and marine habitats. The El Tor biotype of V. cholerae O1 exhibits phase variation between the smooth and rugose morphotypes. Of these, the rugose variant is thought be particularly well adapted for long-term survival in natural water sources: it produces an extracellular polysaccharide (EPS) that enables it to form three-dimensional biofilms and it confers resistance to chlorine and hydrogen peroxide. EPS expression is positively regulated by VpsR, a response regulator of the two-component signal transduction regulator class and negatively regulated by HapR, a homologue of the LuxR regulator of V. harveyi. The main purpose of this proposal is capitalize on the recent publication of the V. cholerae O1 El Tor genome sequence by using microarray expression profiling and bioinformatic tools to capture and analyze the transcriptomes of the smooth and rugose colonial morphotypes and to define the VpsR and HapR regulons. This will greatly facilitate the goals of the parent grant (AI43422, "EPS: structure, regulation and function") and in particular Specific Aim #2 of that grant which focuses on the regulation of EPS expression. Using a whole-genome DNA V. cholerae microarray fabricated in the laboratory of the P.I., expression profiles will be obtained from the smooth and rugose colony types and from hapR and vpsR mutants. Clustering algorithms will be used to identify genes that are differentially regulated in each colony type and genes whose regulation is HapR or VpsR-dependent. A novel bioinformatic tool, BioProspector, will be employed to identify HapR or VpsR consensus sequences in the upstream promoter regions of the genes believed to compose the HapR and VpsR regulons. Gel retardation assays will be performed to corroborate the BioProspector-derived predictions. The possible significance of this project comes from the likelihood that it will provide a deeper understanding of the nature of "rugosity" and thus of the capacity of V. cholerae O1 El Tor to persist in environmental reservoirs. This proposal is consistent with the exploratory/development nature of the R21 mechanism because it explores the combined use of microarray expression profiling and bioinformatics to define regulons--potentially a very powerful, but incompletely examined application of this methodology.